Enzymatic Method for Preparing Rebaudioside M

ABSTRACT

Provided is a method for preparing rebaudioside M by using an enzyme method. In the method, rebaudioside A or rebaudioside D is used as a substrate; and in the existence of a glucosyl donor, rebaudioside M is generated by means of reaction of the substrate under the catalysis of UDP-glucosyl transferase and/or recombinant cells containing the UDP-glucosyl transferase.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name: Sequence Listing.txt; Size: 12,448 bytes; and Date of Creation: Aug. 11, 2017) is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for preparing rebaudioside M, and in particular to a biological method for preparing rebaudioside M.

BACKGROUND ART

Sweetening agents are a class of food additives that have wide applications in the production of food products, beverages and candy. They may be added in the production process of a food product, or alternatively may be used through appropriate dilution as a surrogate for sucrose during household baking. Sweetening agents include natural sweetening agents, for example, sucrose, high fructose corn syrup, honey, etc., and artificial sweetening agents, for example, aspartame, saccharine, etc. Steviosides are a class of natural sweetening agents extracted from the plant Stevia rebaudiana, and are widely used in food products and beverages at present. The extract of Stevia rebaudiana contains a variety of steviosides comprising rebaudioside. Naturally extracted steviosides have great differences in ingredients across different batches, and need subsequent purification. A current commercialized product rebaudioside A comprises some other steviosides, for example, rebaudiosides C, D and F, etc. Stevioside prepared by an extraction method generally further has some impurities mixed therein, which will cause a certain influence on the application field thereof. Rebaudioside M has advantages over rebaudioside A, but has a very low content in leaves of Stevia rebaudiana, and is detected only in a Stevia rebaudiana Morita plant (2010, J. Appl. Glycosci., 57, 199-209). There is yet to be any commercialized production of rebaudioside M at present.

SUMMARY OF THE INVENTION

A technical issue to be solved in the present invention is to provide a method for preparing rebaudioside M by an enzyme method in order to overcome drawbacks in the prior art. This method can produce a high-purity rebaudioside M product at a lower cost within a shorter period of time.

In order to solve the above technical issue, the present invention employs a technical solution as follows: a method for preparing rebaudioside M by an enzyme method. In the method, rebaudioside A or rebaudioside D is used as a substrate; and in the existence of a glucosyl donor, rebaudioside M is generated by means of reaction of the substrate under the catalysis of UDP-glucosyl transferase and/or recombinant cells containing the UDP-glucosyl transferase.

According to the present invention, the glucosyl donor may be UDP-glucose, or a UDP-glucose regeneration system composed of sucrose, sucrose synthetase and UDP (2007, FEBS Letters, 581, 2562-2566), and preferably the UDP-glucose regeneration system composed of sucrose, sucrose synthetase and UDP. UDP glucose is more costly, and the employment of the UDP-glucose regeneration system can substantially reduce the cost.

According to the present invention, the UDP-glucosyl transferase (i.e., uridine diphosphoglucosyl transferase, abbreviated as UGT) is known. Preferably, the UDP-glucosyl transferase employed in the present invention is UGT-A from Stevia rebaudiana and/or UGT-B from Oryza sativa.

The amino acid sequence of UGT-A may have at least 60% identity to sequence 2. Preferably, the amino acid sequence of UGT-A has at least 70% identity to sequence 2. Further preferably, the amino acid sequence of UGT-A has at least 80% identity to sequence 2. Most preferably, the amino acid sequence of UGT-A has at least 90% identity to sequence 2. According to one particular aspect, the amino acid sequence of UGT-A is completely identical to sequence 2.

The amino acid sequence of UGT-B may have at least 60% identity to sequence 4. More preferably, the amino acid sequence of UGT-B has at least 70% identity to sequence 4. Further preferably, the amino acid sequence of UGT-B has at least 80% identity to sequence 4. Most preferably, the amino acid sequence of UGT-B has at least 90% identity to sequence 4. According to one particular aspect, the amino acid sequence of UGT-B is completely identical to sequence 4.

According to the present invention, the reaction may be carried out in an aqueous phase system at a temperature from 4° C. to 50° C. and a pH value from 5.0 to 9.0. Preferably, the reaction is carried out in an aqueous phase system at a temperature from 25° C. to 35° C. and a pH value from 6.5 to 7.5.

More preferably, the reaction is carried out at a temperature of 30° C.

More preferably, the reaction is carried out at a pH value of 7.0.

According to one particular preferred aspect, the reaction is carried out in a phosphate buffer at pH 7.

According to the present invention, when the catalysis is carried out employing recombinant cells containing the UDP-glucosyl transferase, the reaction may be carried out in the presence of a cellular permeating agent. Preferably, the cellular permeating agent is toluene, at a concentration that may be from 1% to 3% according to the ratio by volume in the whole reaction system. More preferably, toluene has a concentration of 2% according to the ratio by volume.

According to the present invention, the recombinant cells may be and are preferably microbial cells, where the microorganisms may be and are preferably Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris, and the like.

According to one particular and preferred aspect, the preparation method is implemented as follows: all the raw materials employed in the reaction are added into a reaction kettle, mixed uniformly, then placed at a set temperature, and stirred for reaction. After completion of the reaction, a rebaudioside M product meeting the use requirements can be obtained through purification treatment. In one particular purification method, a rebaudioside M product with purity as high as 95% can be obtained according to the purification method through aftertreatment including separation by resin.

According to a particular aspect of the present invention, the substrate is rebaudioside A, and the UDP-glucosyl transferase is a mixture of UGT-A from Stevia rebaudiana and UGT-B from Oryza sativa, where the amino acid sequence of UGT-A from Stevia rebaudiana has at least 80% identity to sequence 2, and the amino acid sequence of UGT-B from Oryza sativa has at least 80% identity to sequence 4. Preferably, in the mixture, UGT-A from Stevia rebaudiana and UGT-B from Oryza sativa have a ratio by weight that is 1:0.8 to 1.2, for example, that may be 1:1.

According to yet another particular aspect of the present invention, the substrate is rebaudioside D, and the UDP-glucosyl transferase is UGT-A from Stevia rebaudiana, where the amino acid sequence of UGT-A from Stevia rebaudiana has at least 80% identity to sequence 2.

As a result of implementation of the above technical solutions, the present invention has the following advantages as compared with the prior art.

The method for preparing rebaudioside M by an enzyme method provided in the present invention has an important application value. Because the growth rate of microorganisms is far faster than that of plants, by employing the preparation method according to the present invention, the production cost can be reduced dramatically, the production cycle can be shortened, and the competitive power of the product can be improved greatly. In addition, stevioside has a low content in plants, and has relatively more steviosides of different structures, so that it is extremely difficult to extract purer products. Whereas the employment of the synthetic method by using an enzyme method according to the present invention is capable of providing products with higher purity, which will further expand the application field thereof. As compared with the technique for extracting rebaudioside M from leaves of Stevia rebaudiana, the method according to the present invention possesses a significantly shortened production cycle, improved productive capacity, lower cost, and can provide products with higher purity, and thus can be used more economically in industries of food products and beverages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a proton magnetic spectrum diagram of a product obtained in Example 5 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following rebaudioside A, rebaudioside D, and rebaudioside M are abbreviated respectively as Reb A, Reb D and Reb M, with structural formulae respectively referring to Formulae I, II, and III.

The present invention provides mainly four routes for synthesizing Reb M:

Route 1:

Route 2:

Route 3:

Route 4:

According to the present invention, employed UGT-A or UGT-B may occur in a form of a lyophilized enzyme powder, or present in recombinant cells.

UGT-A or UGT-B is obtained by a method as follows:

Recombinant Escherichia coli (or other microbial bacteria) expression strains of UGT-A or UGT-B are obtained by utilizing molecular cloning techniques and genetic engineering techniques. Then the recombinant Escherichia coli are fermented, to prepare recombinant cells containing UGT-A or UGT-B, or to prepare lyophilized powders of UGT-A or UGT-B.

The above molecular cloning techniques and genetic engineering techniques are all known. Molecular cloning techniques may be seen in Molecular Cloning: A Laboratory Manual. 3rd Edition, by J. Sambrook, 2005.

Expression steps for constructing the recombinant strains of the present invention by employing the genetic engineering technique are as follows:

(1) (according to sequence 1 and sequence 2, or according to sequence 3 and sequence 4) required gene fragments are genetically synthesized, pUC57 vectors are ligated therein, and NdeI and BamHI enzyme cutting sites are added on both ends respectively;

(2) through double enzyme digestion and the ligation, each of the gene fragments is inserted into the corresponding enzyme cutting sites of the expression vector pET30a, to allow each of the genes to be subjected to the control of the T7 promoter; and

(3) recombinant plasmids are transformed into Escherichia coli BL21 (DE3), and target proteins are induced to express by utilizing IPTG, so as to obtain recombinant Escherichia coli expression strains of UGT-A or UGT-B.

The recombinant cells containing UGT-A or UGT-B, or the lyophilized powders of UGT-A or UGT-B are prepared by utilizing the recombinant Escherichia coli expression strains containing UGT-A or UGT-B, by steps as follows:

The recombinant Escherichia coli expression strains containing UGT-A or UGT-B are inoculated into 4 ml of a liquid LB medium at a proportion of 1%, and shaken (200 rpm) at 37° C. for culturing overnight. The culture that has experienced overnight culturing is transferred to 50 ml of the liquid LB medium in an inoculum size of 1%. The culture medium is shaken (200 rpm) at 37° C. for culturing to an OD600 value up to 0.6-0.8. IPTG at a final concentration of 0.4 mM is added therein, and the mixture is shaken at 20° C. for culturing overnight. After completion of the induction, cells are collected by centrifugation (8,000 rpm, 10 min). The cells are resuspended using 5 ml of a 2 mmol/L phosphate buffer (pH 7.0) to obtain recombinant cells, or further ruptured ultrasonically in an ice bath to obtain a lyophilized powder by centrifuging the ruptured liquid (8,000 rpm, 10 min), collecting the supernatant, and lyophilizing for 24 hours.

The present invention will be described below in more details in conjunction with particular examples.

Example 1: Preparation of Recombinant Escherichia coli Cells Containing UGT-A

According to sequence 1 and sequence 2, UGT-A gene fragments were genetically synthesized, NdeI and BamHI enzyme cutting sites were added on both ends respectively, and pUC57 vectors (Suzhou Genewiz Biotech Co., Ltd.) were ligated therein. The UGT gene fragments were subjected to enzyme digestion with restriction endonucleases NdeI and BamHI. Purified fragments were recovered. T4 ligase was added therein, and the fragments were ligated into corresponding enzyme cutting sites of pET30a, to transform the BL21 (DE3) strains.

The UGT strains were inoculated into 4 ml of a liquid LB medium at a proportion of 1%, and shaken (200 rpm) at 37° C. for culturing overnight. The culture that had experienced overnight culturing was transferred to 50 ml of the liquid LB medium in an inoculum size of 1%. The culture medium was shaken (200 rpm) at 37° C. for culturing to an OD₆₀₀ value up to 0.6-0.8. IPTG at a final concentration of 0.4 mM was added therein, and the mixture was shaken at 20° C. for culturing overnight. After completion of the induction, cells were collected by centrifugation (8,000 rpm, 10 min). The cells were resuspended using 5 ml of a 2 mmol/L phosphate buffer (pH 7.0) to obtain recombinant cells containing UGT-A for use in the catalysis.

Example 2: Preparation of Lyophilized UGT-A Powder

The recombinant cells of UGT-A prepared in Example 1 were ruptured ultrasonically in an ice bath, to obtain a lyophilized powder of UGT-A by centrifuging the ruptured liquid (8,000 rpm, 10 min), collecting the supernatant, and lyophilizing for 24 hours.

Example 3: Preparation of Recombinant Escherichia coli Cells Containing UGT-B

According to sequence 3 and sequence 4, UGT-B gene fragments were genetically synthesized, NdeI and BamHI enzyme cutting sites were added on both ends respectively, and pUC57 vectors (Suzhou Genewiz Biotech Co., Ltd.) were ligated therein. The UGT gene fragments were subjected to enzyme digestion with restriction endonucleases NdeI and BamHI. Purified fragments were recovered. T4 ligase was added therein, and the fragments were ligated into corresponding enzyme cutting sites of pET30a, to transform the BL21 (DE3) strains.

The UGT strains were inoculated into 4 ml of a liquid LB medium at a proportion of 1%, and shaken (200 rpm) at 37° C. for culturing overnight. The culture that had experienced overnight culturing was transferred to 50 ml of the liquid LB medium in an inoculum size of 1%. The culture medium was shaken (200 rpm) at 37° C. for culturing to an OD₆₀₀ value up to 0.6-0.8. IPTG at a final concentration of 0.4 mM was added therein, and the mixture was shaken at 20° C. for culturing overnight. After completion of the induction, cells were collected by centrifugation (8,000 rpm, 10 min). The cells were resuspended using 5 ml of a 2 mmol/L phosphate buffer (pH 7.0) to obtain recombinant cells containing UGT-B for use in the catalysis.

Example 4: Preparation of Lyophilized UGT-B Powder

The recombinant cells of UGT-B prepared in Example 3 were ruptured ultrasonically in an ice bath, to obtain a lyophilized powder of UGT-B by centrifuging the ruptured liquid (8,000 rpm, 10 min), collecting the supernatant, and lyophilizing for 24 hours.

Example 5: Synthesis of Reb M by an Enzyme Method with Reb D as a Substrate (Route 1)

In this example, the lyophilized UGT-A powder prepared according to the method in Example 2 was used in the catalytic synthesis of Reb M.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.255 g of UDP glucose, 0.17 g of Reb D, and 1.5 g of the lyophilized UGT-A powder were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution: methanol=20%: 80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb D was more than 40%. 0.054 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, separation by silica gel resin, crystallization, etc.

Example 6: Synthesis of Reb M by an Enzyme Method with Reb a as a Substrate (Route 2)

In this example, the lyophilized UGT-A powder prepared according to the method in Example 2 and the lyophilized UGT-B powder prepared according to the method in Example 4 were used in the catalytic synthesis of Reb M.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.51 g of UDP glucose, 0.145 g of Reb A, and 1.5 g each of lyophilized powders of UGT-A and UGT-B were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution: methanol=20%: 80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb A was more than 40%. 0.05 g of Reb M with a purity greater than 95% was obtained after purification by after treatments, such as, separation by silica gel resin, crystallization, etc.

Example 7: Synthesis of Reb M by an Enzyme Method with Reb D as a Substrate (Route 3)

In this Example, a UDP-glucose regeneration system composed of sucrose, sucrose synthetase (AtSUS1 for short hereafter) from Arabidopsis thaliana, and UDP was used as a glucosyl donor.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.182 g of UDP, 51.3 g of sucrose, 0.17 g of Reb D, 1.5 g of the lyophilized UGT-A powder and 0.5 g of the lyophilized AtSUS1 powder were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution: methanol=20%: 80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb D was more than 80%. 0.11 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, separation by silica gel resin, crystallization, etc.

Example 8: Synthesis of Reb M by an Enzyme Method with Reb a as a Substrate (Route 4)

In this Example, a UDP-glucose regeneration system composed of sucrose, sucrose synthetase (referred to as AtSUS1 hereafter) from Arabidopsis thaliana, and UDP was used as a glucosyl donor.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.364 g of UDP, 51.3 g of sucrose, 0.145 g of Reb A, 1.5 g each of UGT-A and UGT-B, and 0.5 g of the lyophilized AtSUS1 powder were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution: methanol=20%: 80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb A was more than 80%. 0.108 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, separation by silica gel resin, crystallization, etc.

Example 9: Synthesis of Reb M by Whole Cell Catalytic Synthesis with Reb D as a Substrate

In this example, the recombinant cells containing UGT-A prepared according to the method in Example 1 was used in the catalytic synthesis of Reb M.

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.255 g of UDP glucose, 3 mL of toluene, 0.17 g of Reb D, and 10 g of recombinant cells containing UGT-A were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution: methanol=20%: 80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb D was more than 40%. 0.052 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, centrifugation, separation by passing the supernatant through silica gel resin, crystallization, etc.

Example 10: Synthesis of Reb M by Whole Cell Catalytic Synthesis with Reb A as a Substrate

150 mL of a 0.05 mol/L phosphate buffer (pH 7.0), 0.51 g of UDP glucose, 3 mL of toluene, 0.145 g of Reb A, and 10 g of whole cells containing UGT-A and UGT-B at the same time were added successively into the reaction system, mixed uniformly, then placed in a water bath at 30° C., and stirred at 160 rpm to carry out reaction for 2 hours. After completion of the reaction, 500 μl of the reaction solution was taken and added into an equal volume of anhydrous methanol and mixed uniformly. The mixture was centrifuged for 10 min at 8,000 rpm. The supernatant was taken and passed through a filter membrane, followed by detection using high performance liquid chromatography (chromatographic condition: chromatographic column: Agilent eclipse sb-C18 4.6×250 mm; detection wavelength: 210 nm; mobile phase: 1% formic acid aqueous solution: methanol=20%: 80%; flow rate: 1.0 mL/min; column temperature: 25° C.). A conversion rate of Reb A was more than 40%. 0.05 g of Reb M with a purity greater than 95% was obtained after purification by aftertreatments, such as, centrifugation, separation by passing the supernatant through silica gel resin, crystallization, etc.

The above examples are only used for describing technical conception and feature of the present invention, for the purpose of enabling those familiar with the art to understand and thereby implement the content of the present invention, instead of limiting the protection scope of the present invention therewith. Any equivalent changes or modifications made according to the spirit and essence of the present invention shall all be encompassed within the protection scope of the present invention. 

What is claimed is:
 1. A method for preparing rebaudioside M, the method comprising reacting rebaudioside D in a reaction solution with a glucosyl donor in the presence of recombinant cells comprising a UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 2, to obtain rebaudioside M; wherein at least 40% of the rebaudioside D is converted to rebaudioside M after reacting for two hours.
 2. The method of claim 1, wherein the glucosyl donor is UDP-glucose.
 3. The method of claim 2, wherein the UDP-glucose is generated in situ from UDP and sucrose in the presence of a sucrose synthetase.
 4. The method of claim 1, wherein the recombinant cells are microbial cells selected from the group consisting of: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.
 5. The method of claim 1, wherein reacting rebaudioside D with the glucosyl donor in the presence of recombinant cells comprising the UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 2, is carried out in an aqueous phase system having a temperature ranging from 25° C. to 35° C. and a pH ranging from 6.5 to 7.5.
 6. The method of claim 5, wherein the aqueous phase system comprises pH 7 phosphate buffer.
 7. The method of claim 5, wherein the aqueous phase system comprises a cellular permeating agent.
 8. The method of claim 7, wherein the cellular permeating agent is toluene at a concentration of 1% to 3% by volume.
 9. The method of claim 1, wherein the rebaudioside D is prepared by reacting rebaudioside A with a glucosyl donor in the presence of recombinant cells comprising a UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO:
 4. 10. The method of claim 9, wherein the glucosyl donor is UDP-glucose.
 11. The method of claim 10, wherein the UDP-glucose is generated in situ from UDP and sucrose in the presence of a sucrose synthetase.
 12. The method of claim 9, wherein the recombinant cells are microbial cells selected from the group consisting of: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.
 13. The method of claim 9, wherein reacting rebaudioside A with the glucosyl donor in the presence of recombinant cells comprising the UDP-glucosyl transferase having the amino acid sequence of SEQ ID NO: 2, is carried out in an aqueous phase system having a temperature ranging from 25° C. to 35° C. and a pH ranging from 6.5 to 7.5.
 14. The method of claim 13, wherein the aqueous phase system comprises pH 7 phosphate buffer.
 15. The method of claim 13, wherein the aqueous phase system comprises a cellular permeating agent.
 16. The method of claim 15, wherein the cellular permeating agent is toluene at a concentration of 1% to 3% by volume. 